Epoprostenol formulation and method of making thereof

ABSTRACT

This invention relates to a stable epoprostenol composition that can be combined with commercially available IV fluids and can be administered in its reconstituted and/or diluted form under ambient conditions of about 15-30° C. for greater than 24 hours. The composition preferably contains (a) epoprostenol or a salt thereof; (b) a alkalinization agent; and (c) a base, such that when reconstituted or in solution, the solution has a pH&gt;11. Methods for making the lyophilized composition are also disclosed.

This application is a Divisional Application of U.S. Appl. No.12/278,061, filed on Aug. 1, 2008, which application is a United StatesNational Stage Application under 35 U.S.C. 371 of PCT/US07/02948, filedon Feb. 2, 2007, which claims the benefit of U.S. Provisional Appl. Nos.60/764,769, filed Feb. 3, 2006; 60/772,563, filed on Feb. 13, 2006; and60/783,429, filed on Mar. 20, 2006, the contents of each of which areincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a stable epoprostenol compositions that can becombined with commercially available IV fluids for parenteraladministration under ambient conditions of about 15-30° C. for greaterthan 24 hours.

BACKGROUND OF INVENTION

Cardiovascular disorders and diseases, and their associatedcomplications are a principal cause of disabilities and deaths ofindividuals in the United States and Western Europe. For example, inrecent years more than 500,000 deaths have occurred annually in theUnited States alone as a result of coronary artery disease, and anadditional 700,000 patients have been hospitalized for myocardialinfarction.

There has been an ongoing search for effective long term treatment fordisorders and diseases of the heart and arteries, such asatherosclerosis, arteriosclerosis, congestive heart failure, anginapectoris, and other disorders and diseases associated with thecardiovascular system. Prior treatments for such disorders or diseasesinclude administration of vasodilators, angioplasty and by-pass surgery,for example. Such treatments have met with disapproval due to the risksversus the benefits gained by the various treatments. Moreover, suchtreatments have serious shortcomings in long term effectiveness. The useof vasodilators drugs and mechanical treatments for acute and chronicocclusive vascular diseases of the heart, central, and peripheralvascular system have to date been ineffective for favorable long termresults. The outcome with current treatments is minimally impactedbecause the treatments are directed toward the effects of the underlyingdisease process rather than the initial molecular cause of the diseaseor disorder.

For example, the rationale for vasoactive drugs is to reduce bloodpressure by acting directly or indirectly on vascular, and/or cardiac,smooth muscle and thereby decreasing vascular resistance andabnormalities to flow. Such drugs do not treat the initial cause ofelevated pressure and abnormal flow. Rather, they seek to reduce theresulting effect of the disease or disorder. Such drugs activate thesympathetic nervous system by way of the baroreceptor reflex to producean increased heart rate and force of myocardial contraction which arenot necessarily always beneficial effects. Other side effects from suchdrugs include headache, heart palpitations, anxiety, mild depression,dry-mouth, unpleasant taste in the mouth, nausea, vomiting, angina,myocardial infarction, congestive heart failure, decreased cardiacoutput, fluid retention, fatigue, weakness and others. Pharmacologicaltreatment of most diseases is not very specific in its effect on theinitial molecular cause of the disease activity, and treats a verylimited spectrum of effects in diseases which are multi-factorial.

As a further example, such improved outcome in atherosclerotic vasculardiseases is seen with cholesterol reduction and drug treatment for lipiddisorders. However, these treatments do not treat the clottingabnormalities associated with these disease states which are known to bethe proximate event causing heart attack and stroke. These do notprevent the cellular or molecular reactions attributed to platelets,macrophages, neutrophils, lymphocytes, smooth muscle cells, and othercell types known to be involved in atherosclerosis and complications ofthe disease.

Likewise, thrombolytic therapy, angioplasty and by-pass surgery havebeen minimally successful long term. Current mechanical andpharmacological treatments focus on a particular partial or completeocclusion or occluded vessel where, at the particular site, it is eitherunclogged or by-passed with connecting vessels. These treatments fail toaddress the physiologic derangements of normally homeostatic systemswhich allow the occlusive process to begin and progress. Likewise, theyfail to address the multi-centric nature of the homeostaticderangements. These failures frequently result in recurrent occlusion inthe initially treated vessel, and in microemboli from incompleteresolution of thrombus at the occlusive site treated. No treatment isavailable for sites judged to be inadequately occluded or stenotic thatwould respond to currently available, crude technologic methods.

There remains a great need for treatment which prevents the failure ofthe normal homeostatic controls and which restores these controls oncederangements begin to develop. Restoration of the endogenous regulatorysystems and cellular domains to a healthy state could prevent thestenosis, occlusion, thrombosis, and thromboembolic processes whichoccur as a consequence of such derangements. Continuous and episodicrestoration of control in the normal molecular processes which finelyregulate homeostasis can prevent atherosclerosis, variants thereof,hypertension, congestive heart failure, macro and micro-thrombosis andthromboembolism, and complications of these disease processes,including, but not limited to, myocardial infarction, cerebrovascularaccident, related kidney diseases, related central and peripheralnervous system disorders, and related diseases in other cellularsystems. In addition, rapid restoration of homeostatic control onceinjurious processes accelerate and accumulate can minimize both theextent of and duration of consequences on atomic, molecular, membrane,cellular, and organ levels.

Epoprostenol (PGI₂, PGX, prostacyclin), a metabolite of arachidonicacid, is a naturally occurring prostaglandin with potent vasodilatoryactivity and inhibitory activity of platelet aggregation. Epoprostenolis(5Z,9(alpha),11(alpha),13E,15S)-6,9-epoxy-11,15-dihydroxyprosta-5,13-dien-1-oicacid. Epoprostenol sodium has a molecular weight of 374.45 and amolecular formula of C₂₀H₃₁NaO₅, and was approved by the U.S. FDA asFlolan (marketed by GlaxoSmithKline) on Sep. 20, 1995, to treat patientswith cardio obstructive pulmonary disease.

Flolan for Injection is a sterile sodium salt of epoprostenol formulatedfor intravenous (IV) administration. Each lyophilized vial of Flolancontains epoprostenol sodium equivalent to 0.5 mg or 1.5 mgepoprostenol, 3.76 mg glycine, 2.93 mg sodium chloride, and 50 mgmannitol. Sodium hydroxide may also be added to adjust pH.

Flolan is a white to off-white powder that must be reconstituted withsterile diluent for Flolan. Sterile diluent for Flolan is supplied inglass vials containing 94 mg glycine, 73.5 mg sodium chloride, sodiumhydroxide (added to adjust pH) QS to 50 ml Water for Injection, USP. Thereconstituted solution of Flolan has a pH of 10.2 to 10.8 and isincreasingly unstable at lower pH.

Epoprostenol sodium (Formula I), an exocyclic vinyl ether, hydrolyzesrapidly, in a pH dependent fashion, to 6-keto-PGF (Formula II). FormulaI and Formula II are as follows:

The chemical nature, especially the potential hydrolytic lability, ofepoprostenol makes it very difficult to develop a robust formulation.The vinyl ether moiety of PGI₂-Na is best stabilized in solution bybuffering under basic conditions (>pH 8.8). The half-life, time requiredfor 50% lost in potency, of epoprostenol sodium in water as function ofpH is tabulated below in Table 1:

TABLE 1 Solution stability of Epoprostenol in pH 7.2 to 9.3 Temperature(C.) pH Half-life (hours) 0 8.9 21.0 23 8.9 4.4 23 9.3 10.33 23 7.20.033As shown in the above Table 1, 50% of epoprostenol degrades in about 10hours at pH 9.3 at 23° C. In order to manufacture a sterile dosage form,the compound should not lose potency for at least 12 hours preferablyunder ambient conditions. If this is not achievable, the compound mustbe stable at 4° C. for about 12 hours to process under chilledconditions.

Flolan is supplied as a lyophilized vial with a companion vial whichconsists 50 ml of a special diluent buffered with glycine and madeisotonic with sodium chloride. The pH of the isotonic solution isadjusted to a range of 10.2 to 10.8 with sodium hydroxide. Thelyophilized vial is reconstituted with the special diluent andadministered to patients suffering from cardiovascular disorders.

Flolan must be reconstituted only with this sterile diluent for Flolan.Reconstituted solutions of Flolan must not be diluted or administeredwith other parenteral solutions or medications. The reconstitutedsolutions of Flolan must be protected from light and must berefrigerated at 2° to 8° C. (36° to 46° F.) if not used immediately. Therefrigerated solution, however, only lasts two days and must bediscarded thereafter. Additionally, the reconstituted solution cannot befrozen, and the solution must be discarded if it is frozen.

Therefore, there remains a need for epoprostenol formulations that canbe reconstituted with commercially available IV fluids and do notrequire refrigeration after reconstitution until use.

SUMMARY OF THE INVENTION

The present inventor has unexpectedly found that epoprostenol solutionin the presence of an alkalinizing agent, and high pH (>11) is verystable compared to Flolan. Accordingly, one object of the presentinvention is to provide pharmaceutical compositions containingepoprostenol or a salt thereof, and at least one alkalinizing agent atpH>11. The composition is characterized by improved stability uponreconstitution with commercially available intravenous (IV) fluids. Whenreconstituted and/or diluted in commercially available IV fluids, thestability of the present formulation is characterized by at least 90% ofthe original epoprostenol remaining after 24-48 hours at 15-30° C.

Another object of the present invention is to provide methods for makinglyophilized pharmaceutical compositions having epoprostenol and analkalinizing agent. Such a lyophilized composition when reconstitutedhas a pH>11.

Yet another object of the present invention is to provide methods forusing reconstituted lyophilized pharmaceutical compositions havingepoprostenol, and an alkalinizing agent at high pH. The reconstitutedsolution is preferably used to treat cardiovascular diseases, such asatherosclerosis, arteriosclerosis, congestive heart failure, anginapectoris, cardio obstructive pulmonary disease, and hypertension.

Major advantages of the present invention include hemocompatibility andself-preservation (the ability to pass USP preservative effectivenesstest without the presence of preservatives) of the reconstituted and/ordiluted solution. Normally, when a chemical is administeredintravenously, it should be compatible with blood and should not causeblood cell lysis. Generally, high pH formulations and/or hypotonicsolutions cause the lysis of blood cells during the administration.Because the present epoprostenol formulation is administered at high pH(>11), one would expect lysis of the blood cells. However, it wassurprisingly found that blood cell lysis did not occur, and that theepoprostenol solution of the present invention showed the samehemocompatibility as normal saline in our studies. Additionally, thereconstituted and/or diluted solution is highly resistant tomicroorganism and can pass USP preservative effectiveness test.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The composition of the present invention contains epoprostenol, or asalt thereof, and an alkalinizing agent. As used henceforth, the term“epoprostenol” refers to either the free acid or a salt of epoprostenol.The ratio of epoprostenol:alkalinizing agent is preferably about 1:25 toabout 1:200 by weight, more preferably about 1:25 to 1:100, and mostpreferably 1:33.3. Most preferred formulations contain either 0.5 mgepoprostenol and 50 mg of arginine, or 1.5 mg of epoprostenol and 50 mgof arginine per vial. The composition preferably also containssufficient base so that when reconstituted and/or diluted, the pH of thediluted solution is >11.

An alkalinizing agent, as used herein, means an agent that providesalkaline environment (pH>7) when epoprostenol is dissolved in wateralong with the alkalinizing agent. Additionally, although thealkalinizing agent provides an alkaline environment, it does not containa basic hydroxide group, but may contain at least one functional groupthat accepts a proton from water when dissolved in water orwater/organic solvents mixture. The alkalinizing agent should have atleast one pKa greater than 9.0. Preferably, the alkalinizing agent is insolid phase and is soluble in an aqueous medium. The alkalinizing agentsmay be, but are not limited to, arginine, lysine, meglumine, N-methylglucosomine, any other amino acid with a pKa of 9.0 and above, alkalinephosphates such as trisodium phosphates, inorganic carbonates such assodium carbonates, sodium salts of carboxylic acids such astetrasodium-EDTA, or combinations thereof. The most preferredalkalinizing agents are arginine and sodium carbonate.

In certain embodiments, the alkalinizing agent may be common buffersincluding, but not limited to, various salt, acidic, or basic forms ofthe following anions: citrate, phosphate, tartrate, succinate, adipate,maleate, lactate, acetate, bicarbonate, pyruvate, and carbonate.Representative salts of these buffers which may be used are the sodiumand potassium forms, as long as the salt and the amount arephysiologically compatible in an injectable composition. Mixtures ofthese buffering agents may also be used.

The high pH (>11) of the composition (when reconstituted) is preferablyachieved by adding an inorganic base. As used herein, an inorganic baseis defined as a chemical that contains a free hydroxide ion that canspontaneously accept a proton from water and is used for adjusting thepH of the bulk solution to the target value. The preferred inorganicbases are sodium hydroxide, potassium hydroxide, other alkalinehydroxides, divalent hydroxides such as magnesium hydroxide, andvolatile hydroxide such as ammonium hydroxide. Also an organic base,such as primary-, secondary- and tertiary-amines, aromatic amines (suchas aniline), and aromatic alcohol (such as phenol) can be used. Acombination of both organic and inorganic bases are also appropriate forthe present invention. Preferably, the base is added so that the pH ofthe bulk solution is greater than 11, preferably greater than 12, and,most preferably greater than 13. The preferred base for use with thepresent invention is sodium hydroxide.

The composition is preferably a lyophile produced by freeze drying(lyophilizing) a bulk solution containing epoprostenol, or a saltthereof, and arginine. The pH of the bulk solution is preferablyadjusted to about 12.5-13.5, most preferably 13, by the addition ofsodium hydroxide.

The term “lyophilize” with regard to the current pharmaceuticalformulations is intended to refer to freeze drying under reducedpressure of a plurality of vials, each containing a unit dose of theepoprostenol formulation of the present invention therein. Lyophilizers,which perform the above described lyophilization, are commerciallyavailable and readily operable by those skilled in the art. In oneembodiment of the present invention, the bulk solution is lyophilized. Apreferred lyophilization process contains three cycles: a freeze cycle,a primary drying cycle, and a secondary drying cycle. The freeze cyclecomprises the following steps:

-   1. Cooling the shelf to about −30° C. or below at the rate of    approximately 0.5 to 0.7° C./min. and holding the shelf at this    temperature for about 30 to 45 min. or until the product temperature    reaches about −25° C. or below-   2. Lowering the shelf temperature to about −45° C.±2° C. or below    until the product temperature reaches approximately −38±2° C. or    less.-   3. Holding the product at this temperature for approximately six    hours or longer.-   4. Applying vacuum until the chamber pressure reaches in the range    of 50 milliTorr or less-   5. Keeping the shelf temperature at about −45±2° C. for about 45    minutes or more even after vacuum application.

After the freeze cycle, the product is dried in a primary drying cycle,which includes the following steps:

-   1. Raising the shelf temperature to around 0° C.±2° C. at the    heating rate of about 20±2° C./hour, while under vacuum, and    continue drying until the product temperature reaches approximately    −3±2° C. or higher.-   2. Raising the shelf temperature to about 25±2° C. and continue the    drying cycle, while under vacuum, continue drying until the product    temperature reaches about 20±2° C. or higher.

After the primary drying cycle, the product is further dried undervacuum in a secondary drying cycle by increasing the shelf temperatureto approximately 45±2° C. at a rate of about 3±2° C./hr and continuedrying till the product reaches about 38±2° C. or higher. Here,preferably, the drying rate is set very slow such that the time taken toreach about 40±2° C. from about 25±2° C. is about 5 hours.

Other pharmaceutically acceptable excipients may also be used in thecomposition. These excipients may include, but are not limited to,preservatives (present at about 0.1-0.5%), carriers (present at about1-5%), tonicity modifying agents (sufficient amount to make the solutionisotonic), bulking agents (present at about 1-10%), and otherconventional components used in formulating pharmaceutical compositions.Preferably, these excipients do not materially affect the fundamentalcharacteristics of the formulation.

Particular preservatives contemplated for use may include benzylalcohol, parabens, phenol, phenol derivatives, benzalkonium chloride andmixtures thereof. Depending on the particular preservative utilized, theamount of preservative could vary. Preferably the preservative ispresent at about 0.1-0.5%, most preferably 0.2%.

Representative examples of tonicity modifying agents include sodiumchloride, mannitol, dextrose, glucose, lactose and sucrose. The amountof the tonicity modifying agent should be sufficient to render thesolution isotonic. This amount varies with the solution and the type oftonicity modifying agent. However, one skilled in the art would be ableto determine the amount of tonicity modifying agent to render aparticular solution isotonic.

Representative examples of bulking agents include, but are not limitedto, hydroxyl ethyl starch (HES); sugars, such as sorbitol, lactose,dextran, maltose, mannose, ribose, sucrose, mannitol, trehalose,lactose, dextran, cyclodextrin; other mono- or polysaccharides; glycine;polyvinylpyrrolidine (PVP); or combinations thereof. The bulking agentmay be present at about 1-10%, preferably 1-5%, and most preferably 5%.

In a preferred embodiment, the stable lyophilized formulation containsepoprostenol (or a salt thereof, such as epoprostenol sodium), mannitol,and arginine. The ratio of epoprostenol:arginine is about 1:25 to about1:200, more preferably about 1:25 to about 1:100, and most preferablyabout 1:33.3. The ratio of arginine:mannitol is about 5:1 to about 1:5,preferably about 3:1 to about 1:3, and most preferably about 1:1.Preferred formulations contain either 0.5 mg epoprostenol and 50 mg eachof arginine and mannitol or 1.5 mg of epoprostenol and 50 mg each ofarginine and mannitol per vial. The bulk solution for lyophilizationcontains either 0.5 mg epoprostenol and 50 mg each of mannitol andarginine, or 1.5 mg of epoprostenol and 50 mg each of arginine andmannitol per ml. The pH of the bulk solution is adjusted to >11 withsodium hydroxide prior to lyophilization.

In another embodiment, the composition of the present compositioncontains epoprostenol (or a salt thereof, such as epoprostenol sodium),and arginine. The composition may also include a base, which may be aninorganic base, such as sodium hydroxide, or an organic base, orcombination of both organic and inorganic base. The base is added sothat the pH of the bulk solution is greater than 11, preferably greaterthan 12, and, most preferably 13 or higher.

In another embodiment, the present invention developed a stablelyophilized formulation containing epoprostenol (or a salt thereof, suchas epoprostenol sodium), mannitol, and a base, preferably in a ratio ofabout 1:25 to about 1:200 (epoprostenol:mannitol), more preferably 1:100and most preferably 1:33.3. Preferred formulations contain either 0.5 mgepoprostenol and 50 mg of mannitol or 1.5 mg of epoprostenol and 50 mgmannitol per vial. The bulk solution for lyophilization contains eitherboth 0.5 mg epoprostenol and 50 mg of mannitol or 1.5 mg of epoprostenoland 50 mg mannitol per ml. The pH of the bulk solution is adjusted to13.0 with the base.

The lyophilized composition may be reconstituted using commerciallyavailable IV fluids. These fluids include, but are not limited to, waterfor injection (WFI), including bacteriostatic WFI and sterile WFI; 0.9%sodium chloride solution (normal saline); lactated Ringer's solution;Ringer's solution; sodium carbonate solution; bicarbonate solution;amino acid solution; and similar readily available pharmaceuticaldiluents. The preferred diluent is normal saline or lactated Ringer'ssolution. When reconstituted and/or diluted, the pH of the reconstitutedsolution is greater than about 11, preferably greater than about 11.3,more preferably greater than about 11.5, and most preferably greaterthan about 11.8.

The pharmaceutical composition of the present invention is formulated ina unit dose or in multi-dose form, and may be in an injectable orinfusible form such as solution, suspension, or emulsion. Preferably, itis prepared as dried, lyophilized powder, which can be reconstitutedinto the liquid solution, suspension, or emulsion before administrationby any of various methods including'IV routes of administration.Preferably, the lyophilized composition is reconstituted to 100-10μg/ml, preferably 10 μg/ml for administration. This diluted solution is90% stable (90% of the original epoprostenol remains) at 15-30° C. after24-48 hrs.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following examples aregiven to illustrate the present invention. It should be understood thatthe invention is not to be limited to the specific conditions or detailsdescribed in these examples.

EXAMPLE 1 Stability of Flolan for Injection

In order to understand the stability of a version of epoprostenolcurrently available on the market (Flolan), we have prepared lyophilizedvials of epoprostenol as well as the diluent according to thecomposition given in the Physician's Desk Reference (PDR). Flolan forInjection is a sterile sodium salt formulated for intravenous (IV)administration. Each lyophilized vial of Flolan contains epoprostenolsodium equivalent to 0.5 mg or 1.5 mg epoprostenol, 3.76 mg glycine,2.93 mg sodium chloride, and 50 mg mannitol. Sodium hydroxide may havebeen added to adjust pH. We prepared our Flolan simulated products usingthis formula.

Flolan must be reconstituted with sterile diluent made specifically forFlolan. Sterile diluent for Flolan is supplied in glass vials containing94 mg glycine, 73.5 mg sodium chloride, sodium hydroxide (added toadjust pH) QS to 50 ml Water for Injection, USP. The diluent pH rangelisted in the PDR is 10.2 to 10.8, hence we prepared the diluent asabove and adjusted the pH of the diluent to 10.5. Our vials of simulatedproduct were reconstituted with the diluent per the instructions givenin the PDR and the stability of the diluent was monitored 5±1° C. Thestability data is summarized in the Table 2 below. The PDR alsodescribes that the diluted solution must be administered at <25° C.Since the drug is continuously infused via an infusion pump, thesolution pouch is usually kept in an ice pack which needs to be changedevery 8 hours.

TABLE 2 Solution stability of Flolan formulation at 5 ± 1° C., pH 10.5TIME % Assay of Area % of 6-keto PGF (HRS) Epoprostenol by Area et al.impurities Initial 100.0 0.21 2.5 99.7 0.31 5.0 99.3 0.39 7.5 98.9 0.4410.0 98.5 0.55 12.5 98.0 0.61 15.0 97.5 0.71 18.0 97.1 0.80 39.0 86.74.49 53.0 80.3 6.59 77.0 61.6 12.9

As shown in Table 2, the product degrades at a rate of approximately 0.5to 1% for every three hours in the first 39 hours; therefore, in 24hours it degrades about 4-8%. Later times show an even fasterdegradation rate.

We have also conducted the solution stability of the Flolan formulationat 29±1° C. The Flolan formulation degraded just over 4% in 1 hour(presented in Table 3) while a formulation of this present inventionlost of just over 2% of drug in 24 hours (presented in Table 7).

TABLE 3 Solution stability of Flolan formulation pH 10.5, at 29 ± 1° C.TIME % ASSAY of Area % of 6-keto PGF (HRS) Epoprostenol by Area et al.impurities Initial 100.0 0.08 1 95.6 1.48 2 91.2 2.54 3 87.1 3.54 4 83.44.41 5 80.4 5.39

EXAMPLE 2 Stability of Epoprostenol with Arginine

A solution of epoprostenol and 50 mg/ml of arginine was prepared and thestability of this solution at 5° C. was determined. The resulting dataare presented in the Table 4:

TABLE 4 Solution stability of Epoprostenol in presence of 50 mg/ml ofarginine, pH 11.9, at 5 ± 1° C. TIME % Assay of Area % of 6-keto (HRS)Epoprostenol by Area et al. impurities Initial 100.0 0.15 2 99.9 0.19 499.8 0.19 6 99.8 0.21 8 99.7 0.22 10 99.6 0.23 12.5 99.6 0.25 14.5 99.60.26 39 99.5 0.63 53 98.6 1.15 77 92.7 2.05 100 92.5 2.10 124 92.5 2.25

As shown in the Table 4, the composition lost only 1.4% in potency at 53hours, while the Flolan formulation (Table 2) showed approximately 20%potency loss during this time. The data suggest that epoprostenolsolution could be continuously administered for 5 days without changingthe solution in the reservoir, assuming sufficient volume and sterilityare assured. This is a significant improvement over the Flolan becausethe Flolan solution in the pump reservoir needs to be replaced every 12hours.

Stability of the same formulation was also conducted at pH 11.2; and thedata are summarized in the Table 5 below:

TABLE 5 Solution stability of Epoprostenol in presence of 50 mg/ml ofarginine, pH 11.2, at 5 ± 1° C. TIME % Assay of Area % of 6-Keto (HRS)Epoprostenol by Area et al. impurities Initial 100.0 — 4.0 99.5 0.28 7.099.0 0.39 10.0 98.6 0.53 11.5 98.3 0.57 17.5 97.3 0.80 Even at pH 11.2,the data suggest better stability than the Flolan formulation (Table 2).

EXAMPLE 3 Stability of Reconstituted Lyophile

In the next set of experiments, the pH of the solution containingepoprostenol and arginine was adjusted to 13.0 with sodium hydroxide,and lyophilized. Upon reconstitution of the lyophile with 1 ml of Waterfor Injection, the reconstituted solution contains 50 mg/ml arginine and0.5 mg/ml epoprostenol. The pH of the solution is 13.0. The stabilitydata are presented in Table 6 for 5° C. and Table 7 for 29° C. below:

TABLE 6 Solution stability of Epoprostenol in presence of 50 mg/mlarginine at 5 ± 1° C., pH 13.0 TIME % Assay of Area % of 6-keto PGF(Hours) Epoprostenol by Area et al. impurities Initial 100.0 0.16 14.599.8 0.20 53 98.8 0.29 124 98.0 0.40 148 97.6 0.45 192 97.3 0.47 24096.9 0.61 480 96.6 0.70

The current invention therefore shows only 3.4% loss of potency over 480hours, or 0.007%/hour on average when held at 5° C.

In addition, the advantage with the present invention is that theformulation does not require a special diluent. The lyophilizedformulation can be reconstituted with water for injection to aconcentration as low as 5 ng/ml and the pH of the solution is stillmaintained above 11.0 due to buffer capacity of arginine with basicpK_(a) of 13.2 and 10.8 and the additional base added for the pHadjustment.

TABLE 7 Solution stability of Epoprostenol in presence of 50 mg/ml ofarginine, pH 13.0, at 29 ± 1° C. TIME % ASSAY of Area % of 6-keto (HRS)Epoprostenol by Area et al. impurities Initial 100.0 0.072 1 100.0 0.0875 99.8 0.18 7 99.3 0.21 8 99.2 0.25 9 99.1 0.29 10 98.8 0.28 11 98.60.29 12 98.5 0.30 13 98.1 0.38 14 98.0 0.37 15 97.8 0.39 24 97.5 0.39 3697.5 0.45

Finally, because only 1.5% degradation was observed in 12 hours at 29°C., it is conceivable to manufacture this formulation in a parenteralfacility without cooling the bulk solution to 5° C. This would not bepossible for the currently available product because the pH of the bulksolution is 10.5 and the product would have to be manufactured at 5±1°C. within 12 hours or significant degradation is occurs.

EXAMPLE 4 Comparison of Various Epoprostenol Compositions

In the next stage of development, we screened several lyophilizedformulations with the pH of bulk solution for lyophilization adjustedbetween 10.5 and 13.0 in the presence of different excipients. Thecomposition of the studies formulations are detailed in Table 8 and thestability data are summarized in the Table 9 below.

TABLE 8 Stability of several Epoprostenol prototype formulationsQuantity (mg) of Excipient used in Formulations Batch # EPP TrehaloseArginine Mannitol HES NaCl Glycine Na2CO3 Bulk. Sol. pH EPP-7 0.5 50 13EPP-8 0.5 50 3 3.75 10.5 EPP-10 0.5 50 50 13 EPP-12 0.5 100 13 EPP-130.5 50 50 13 EPP-14 0.5 50 13 EPP-19 0.5 50 12 EPP-20 0.5 50 13 EPP-230.5 50 50 13 EPP-24 0.5 50 50 11 EPP-25 0.5 50 50 12 EPP-26 0.5 50 50 13EPP-27 0.5 50 12 EPP-30 0.5 100 97.76 11 EPP-31 0.5 100 97.76 12 EPP-320.5 50 97.76 11 EPP-33 0.5 50 12 EPP-38 0.5 50 13 EPP: epoprostenolsodium; HES: Hydroxy ethyl starch; Bulk. Sol. pH: bulk solution pH

TABLE 9 Stability of Epoprostenol prototype formulations Stability (%Initial) stored at 40° C. Batch # 15 Days 30 Days 60 Days 90 Days EPP-799 97 NP NP EPP-8 40  0 NP NP EPP-10 99 99 99 100  EPP-12 76 NP NP NPEPP-13 99 98 99 97 EPP-14 100 96 97 83 EPP-19(25%)* 87 NP NP NPEPP-20(40%) 29 NP NP NP EPP-23 94 96 EPP-24 0 EPP-25 60 35 24EPP-26(11%) 100 101  100  EPP-27 60 EPP-30 88 EPP-31 90 96 EPP-32 76 74EPP-33 95 100  EPP-38(13%) 94 *The numbers in parenthesis denote thewater content of the lyophile NP: Not performed

During the lyophilization several batches were lyophilized togetherresulting in different moisture contents. The moisture contents ofselected samples (EPP-19, 20. 26, and 38) were also measured. As shownin the Table 8 above, the stability of epoprostenol is better at pH 13compared to lower pH samples. Formulations containing mannitol/HES ormannitol/arginine or HES/sodium carbonate showed excellent stability.

In the next step, formulations containing arginine/mannitol, with the pHof bulk solution adjusted to 13, were selected for lyophilization. Sincemoisture content varies from batch to batch, the lyophilization cyclewas optimized to consistently produce moisture contents less than 12%,using the three cycle lyophilization process discussed above. Using theoptimized lyophilization process, the following formulations weremanufactured:

-   1. Three batches of epoprostenol (0.5 mg)/arginine (50 mg)/mannitol    (50 mg)/pH 13 per vial-   2. One batch of epoprostenol (0.5 mg)/arginine (50 mg)/mannitol (50    mg)/pH 12 per vial-   3. Two batches of epoprostenol (0.5 mg)/arginine (50 mg)/trehalose    (50 mg)/pH 13 per vial-   4. One batch of epoprostenol (0.5 mg)/arginine (50 mg)/trehalose (50    mg)/pH 12 per vial-   5. One batch each of the Flolan composition adjusted to pH 12 and    13.    The moisture content of each of these batches ranged between between    7-10%.

Three-month solid state stability data for the selected formulations arepresented in the Tables 10-18 below:

TABLE 10 Batch # EX-01: EPP/mannitol/Arginine/pH::0.5/50/50/13*Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-PGF Area % 40° C. Initial 0.49 100 Nil Nil NA 15 days 0.49 100Nil Nil NA 1 month 0.50 102 Nil Nil 0.2  2 months 0.48 98 0.003 0.650.72 3 months 0.48 98 0.002 0.49 0.78 25° C. 3 months 0.48 98 0.000340.07 0.12 *EPP/mannitol/arginine/pH::0.5/50/50/13 = 0.5 mg/vialepoprostenol, 50 mg/vial mannitol, 50 mg/vial arginine, and pH 13.

TABLE 11 Batch # EX-02: EPP/mannitol/Arginine/pH::0.5/50/50/13Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-PGF Area % 40° C. Initial 0.49 100 Nil Nil NA 15 days 0.49 100Nil Nil NA 1 month 0.50 102 Nil Nil 0.2  2 months 0.48 98 0.003  0.640.73 3 months 0.49 100 0.0041 0.84 0.87 25° C. 3 months 0.49 100 0.00040.08 0.12

TABLE 12 Batch # EX-03: EPP/mannitol/Arginine/pH::0.5/50/50/13Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-PGF Area % 40° C. Initial 0.49 100 Nil Nil NA 15 days 0.49 100Nil Nil NA 1 month 0.50 102 Nil Nil 0.2  2 months 0.50 102 0.0021 0.410.66 3 months 0.48 98 0.0041 0.84 1.07 25° C. 3 months 0.49 100  0.000440.09 0.12

TABLE 13 Batch # EX-07: EPP/mannitol/Arginine/pH::0.5/50/50/12Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-keto Area % 40° C. Initial 0.47 100 0.002 0.36 0.08 15 days0.013 2.8 0.07 14.8 0.76 1 month 0.008 1.7 0.072 15.4 0.66

TABLE 14 Batch # EX-04: EPP/trehalose/Arginine/pH::0.5/50/50/13Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-PGF Area % 40° C. Initial 0.52 100 0.0006 0.11 0.08 15 days0.52 100 0.0007 0.13 0.08 1 month 0.52 100 NIL NIL 0.12 2 months 0.49 940.007  1.4  0.38 3 months 0.49 94 0.0114 2.2  0.80 25° C. 3 months 0.52100 0.0006 0.12 0.17

TABLE 15 Batch # EX-06: EPP/Arginine/Trehalose/pH::0.5/50/50/13Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial 6-PGF Area % 40° C. Initial 0.52 100 0.0006 0.12 0.08 15 days0.52 100 0.0006 0.12 0.08 1 month 0.50 96 NIL NIL 0.11 2 months 0.48 920.011  2.08 0.31 3 months 0.49 94 0.012  2.3  0.87 25° C. 3 months 0.5198 0.0001 0.02 0.17

TABLE 16 Batch # EX-05: EPP/trehalose/Arginine/pH::0.5/50/50/12Additional Storage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPPmg/vial EPP Area % 40° C. Initial 0.51 100.0 Nil NIL 0.15 15 days 0.51100.0 0.003 0.57 0.2 1 month 0.40 78 0.003 0.74 0.45 2 months 0.32 630.004 0.82 1.33

TABLE 17 Batch # EX-08: Flolan simulated formulation*: pH 12 AdditionalStorage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial Initialmg/vial EPP Area % 40° C. Initial 0.50 100.0 0.0014 0.28 0.11 15 days0.03 6.0 0.072 14.4 1.56 1 month 0.011 2.2 0.032 6.4 0.78 *Flolansimulation formulation refers to a formulation that is identical to thecommercially available Flolan marketed by GlaxoSmithKline, except thatthe pH has been adjusted to the indicated pH.

TABLE 18 Batch # EX-09: Flolan simulated formulation: pH 13 AdditionalStorage Assay EPP % of 6-PGF % of Peaks Temp Time mg/vial EPP mg/vial6-PGF Area % 40° C. Initial 0.50 100 0.001 0.2 0.12 15 days 0.50 1000.0012 0.24 0.12 2 months 0.44 88 0.003 0.6 0.82 3 months 0.45 90 0.00831.7 0.3 25° C. 3 months 0.48 96 0.00074 0.15 NIL

As seen from the data above, epoprostenol is most stable inmannitol/arginine containing formulations when the pH of the bulksolution adjusted to 13. This is followed by arginine/trehaloseformulations with the bulk solution for lyophilization adjusted to pH13. Either trehalose or mannitol formulations with arginine at lower pHconditions are less stable at 40° C. compared to the pH 13 formulations.The simulated lyophilized Flolan formulation degraded almost completelyat one month/40° C. at pH 12. At pH 13 it showed a better stability, butnot as good as the mannitol/arginine/pH 13 formulation.

EXAMPLE 5 Stability of Various Reconstituted Epoprostenol Diluted to 10μg/ml

Dilution studies were also conducted to determine whether theformulations of the present invention are suitable for IV infusion atroom temperature. The stability studies were conducted at 25° C. and 30°C. to mimic the temperatures during the infusion over a 24 hour periodin various large volume parenteral solutions.

To this end, the stability of the epoprostenol lyophile reconstitutedand diluted to 10 μg/ml in normal saline was stability monitored for 48hours at 25° C. and 30° C. Dilution stability of all three primaryformulation batches was conducted in normal saline at 25° C. and 30° C.In addition to these studies, dilution stability studies on one lot ofprimary formulation batch were conducted at 25° C. and 30° C. in 5%Dextrose (D5W), WFI (in-house) and lactated Ringer's solution.

For the dilution studies, each vial was reconstituted with 5 ml of thediluent. The clear solution was transferred into a 50 ml volumetricflask. The vial was rinsed with 5 ml of diluent three times and therinses were transferred to the flask. The contents of the flask werefurther diluted with the diluent and made up to the mark with thediluent. The pH of the diluted solution was measured and recorded. Thecontents of the flask were held at the temperatures noted and analyzedat predetermined time intervals. The dilution stability data in variousdiluents are presented in the Tables 19-30 below:

Dilution Studies in Normal Saline

TABLE 19 Dilution stability of Epoprostenol in Saline, Lot # EX-01 at25° C., pH 11.58 Additional Assay EPP % of 6-PGF* % of Peaks Time(ug/ml) Initial (ug/ml) EPP(Initial) Area % Initial 10.10 100.0 NIL NILNIL  6 hrs 10.06 99.6 NIL NIL NIL 12 hrs 10.02 99.2 NIL NIL NIL 18 hrs9.95 98.5 NIL NIL NIL 24 hrs 9.81 97.1 0.12 1.19 NIL 30 hrs 9.71 96.10.32 3.17 NIL 36 hrs 9.62 95.2 0.40 3.96 NIL 42 hrs 9.52 94.3 0.48 4.75NIL 48 hrs 9.46 93.7 0.52 5.14 NIL *6-PGF—6-keto PGF

TABLE 20 Dilution stability of Epoprostenol in Saline, Lot # EX-01 at30° C. Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml) Initial(ug/ml) EPP(Initial) Area % Initial 10.10 100.0 NIL NIL NIL  6 hrs 10.0599.5 NIL NIL NIL 12 hrs 9.92 98.2 NIL NIL NIL 18 hrs 9.79 96.9 0.37 3.65NIL 24 hrs 9.62 95.3 0.59 5.89 NIL 30 hrs 9.37 92.8 0.76 7.52 NIL 36 hrs9.21 91.2 0.83 8.22 NIL 42 hrs 9.02 89.3 1.30 12.87 NIL 48 hrs 8.94 88.51.34 13.27 NIL

TABLE 21 Dilution stability of Epoprostenol in Saline, Lot # EX-02 at25° C., pH 11.58 Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml)Initial (ug/ml) EPP(Initial) Area % Initial 10.30 100.0 NIL NIL NIL  6hrs 10.24 99.4 NIL NIL NIL 12 hrs 10.20 99.0 NIL NIL NIL 18 hrs 10.0097.1 NIL NIL NIL 24 hrs 9.96 96.7 0.07 0.68 NIL 30 hrs 9.85 95.6 0.272.62 NIL 36 hrs 9.76 94.8 0.34 3.30 NIL 42 hrs 9.68 94.0 0.44 4.27 NIL48 hrs 9.58 93.0 0.48 4.66 NIL

TABLE 22 Dilution stability of Epoprostenol in Saline, Lot # EX-02 at30° C. Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml) Initial(ug/ml) EPP(Initial) Area % Initial 10.20 100.0 NIL NIL NIL  6 hrs 10.1399.3 NIL NIL NIL 12 hrs 10.03 98.3 NIL NIL NIL 18 hrs 9.82 96.3 0.323.14 NIL 24 hrs 9.70 95.1 0.52 5.10 NIL 30 hrs 9.47 92.8 0.70 6.90 NIL36 hrs 9.29 91.1 0.79 7.74 NIL 42 hrs 9.10 89.2 1.21 11.86 NIL 48 hrs9.02 88.4 1.27 12.45 NIL

TABLE 23 Dilution stability of Epoprostenol in Saline, Lot # EX-03 at25° C., pH 11.6 Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml)Initial (ug/ml) EPP(Initial) Area % Initial 10.30 100.0 NIL NIL NIL  6hrs 10.20 99.0 NIL NIL NIL 12 hrs 10.20 99.0 NIL NIL NIL 18 hrs 10.0097.1 NIL NIL NIL 24 hrs 9.94 96.5 0.09 0.87 NIL 30 hrs 9.82 95.3 0.292.81 NIL 36 hrs 9.71 94.3 0.37 3.59 NIL 42 hrs 9.61 93.3 0.46 4.47 NIL48 hrs 9.53 92.5 0.51 4.95 NIL

TABLE 24 Dilution stability of Epoprostenol in Saline, Lot # EX-03 at30° C. Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml) Initial(ug/ml) EPP(Initial) Area % Initial 10.20 100.0 NIL NIL NIL  6 hrs 10.1099.0 NIL NIL NIL 12 hrs 9.96 97.6 NIL NIL NIL 18 hrs 9.77 95.8 0.33 3.24NIL 24 hrs 9.61 94.2 0.58 5.69 NIL 30 hrs 9.44 92.5 0.75 7.35 NIL 36 hrs9.30 91.2 0.83 8.14 NIL 42 hrs 9.10 89.2 1.30 12.75 NIL 48 hrs 8.96 87.81.33 13.04 NIL

As shown in Tables 19-24, the diluted solutions of epoprostenol werequite stable at 25° C. and 30° C. maintaining greater than 90% potencyfor at least a 24 hour period. All batches studied exhibited minimalbatch to batch variability in stability at both temperatures. The onlydegradation product observed was 6-keto PGF.

Dilution Studies in D5W:

TABLE 25 Dilution stability of Epoprostenol in D5W, Lot # EX-03 at 25°C., pH 10.9 Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml)Initial (ug/ml) EPP(Initial) Area % Initial 10.20 100.0 NIL NIL NIL 2hrs 9.83 96.4 NIL NIL NIL 4 hrs 9.40 92.2 0.03 0.29 NIL 6 hrs 9.08 89.00.04 0.39 NIL 8 hrs 8.81 86.4 0.07 0.69 NIL

TABLE 26 Dilution stability of Epoprostenol in D5W, Lot # EX-03 at 30°C. Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml) Initial(ug/ml) EPP(Initial) Area % Initial 10.20 100.0 NIL NIL NIL 2 hrs 9.7695.7 NIL NIL NIL 4 hrs 9.40 92.2 0.04 0.39 NIL 6 hrs 9.04 88.6 0.04 0.39NIL 8 hrs 8.60 84.3 0.08 0.80 NIL

The epoprostenol degraded in 5% Dextrose solution (D5W) more than in thesaline. The 6-keto PGF levels were very low, yet no other peaks wereobserved. Here, approximately 84% of the drug degraded after 8 hours,but no other peaks were detected as a degradation product.

The instability in D5W can be partially attributed to the significantdrop in the pH, as the pH drop was more than expected. In the case ofsuch a pH drop, D5W cannot be used for reconstitution/dilution of thispresent invention.

Dilution Stability Study of Epoprostenol in Water for Injection

TABLE 27 Dilution stability of Epoprostenol in WFI, Lot # EX-03 at 25°C., pH 11.55 Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml)Initial (ug/ml) EPP(Initial) Area % Initial 9.04 100.0 NIL NIL NIL  6hrs 8.97 99.2 NIL NIL NIL 12 hrs 8.86 98.0 NIL NIL NIL 18 hrs 8.77 97.0NIL NIL NIL 24 hrs 8.68 96.0 0.11 1.20 NIL 30 hrs 8.60 95.1 0.12 1.30NIL 36 hrs 8.60 95.0 0.41 4.54 NIL 42 hrs 8.43 93.3 0.46 5.10 NIL 48 hrs8.41 93.0 0.80 8.90 NIL

TABLE 28 Dilution stability of Epoprostenol in WFI, Lot # EX-03 at 30°C. Assay EPP % of 6-PGF % of Additional Peaks Time (ug/ml) Initial(ug/ml) EPP(Initial) Area % Initial 9.04 100.0 NIL NIL NIL  6 hrs 8.9398.8 0.06 0.7 NIL 12 hrs 8.78 97.1 0.09 1.04 NIL 18 hrs 8.25 91.3 0.212.30 NIL 24 hrs 7.27 80.4 0.48 5.32 NIL 30 hrs 5.75 64.0 0.78 8.60 NIL36 hrs 3.37 37.3 1.76 19.5 NIL 42 hrs 1.64 18.1 3.20 35.0 21.1 48 hrs0.79 8.73 4.30 47.2 26.2

Interestingly the stability of epoprostenol in water and normal salineat 25° C. were similar. However, epoprostenol in water degraded morerapidly at 30° C. than in normal saline. However, greater than 90%potency was maintained for more than 18 hours. Degradation acceleratedafter the 24 hours time point.

Dilution Stability Study in Lactated Ringer's Solution

Dilution stability in lactated Ringer's solution has also been conductedand shown in Tables 29-30 below:

TABLE 29 Dilution stability of Epoprostenol in Lactated Ringer'ssolution, Lot # EX-03 at 25° C., pH 11.63 Assay EPP % of 6-PGF % ofAdditional Peaks Time (ug/ml) Initial (ug/ml) EPP(Initial) Area %Initial 10.50 100.0 NIL NIL NIL  6 hrs 10.43 99.3 0.17 1.6 NIL 12 hrs10.20 97.1 0.24 2.3 0.9 18 hrs 10.08 96.0 0.25 2.4 2.0 24 hrs 9.98 95.10.17 1.6 3.6 30 hrs 9.94 94.7 0.19 1.8 3.5 36 hrs 9.82 93.5 0.19 1.8 3.442 hrs 9.74 92.8 0.18 1.7 3.2 48 hrs 9.61 91.5 0.35 3.3 3.1

TABLE 30 Dilution stability of Epoprostenol in Lactated Ringer'ssolution, Lot # EX-03 at 30° C. Assay EPP % of 6-PGF % of AdditionalPeaks Time (ug/ml) Initial (ug/ml) EPP(Initial) Area % Initial 10.50100.0 NIL NIL NIL  6 hrs 10.34 98.5 0.09 0.83 2.59 12 hrs 10.31 98.20.13 1.22 3.85 18 hrs 10.20 97.1 0.09 0.87 6.04 24 hrs 9.82 93.5 0.111.00 6.00 30 hrs 9.62 91.6 0.15 1.40 1.04 5.76 42 hrs 9.26 88.2 0.181.72 6.24

The stability of epoprostenol in the lactated Ringer's solution iscomparable to that of normal saline at both temperatures studied.

Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

What is claimed is:
 1. A method for making an epoprostenol compositioncomprising the steps of: (a) providing a bulk solution comprising (i)epoprostenol or a salt thereof, and (ii) an alkalinizing agent; and (b)adjusting the pH of the bulk solution to greater than
 13. 2. The methodof claim 1, further comprising the step of: (c) lyophilizing theadjusted bulk solution.
 3. The method of claim 2, wherein step (c)comprises: freezing the adjusted bulk solution in a freezing cycle;drying the frozen solution in a primary drying cycle; and drying thefrozen solution in a secondary drying cycle.
 4. The method of claim 3,wherein the freezing cycle comprises: (i) placing the bulk solution on ashelf in a lyophilization chamber; (ii) cooling the shelf to less thanor about −30 degrees C. at the rate of approximately 0.5 to 0.7 C./min.;(iii) holding the shelf at about −30 degrees C. or below for about 30min or until the adjusted bulk solution temperature reaches less than orabout −25 degrees C.; (iv) lowering the shelf temperature to about −45±2degrees C. until the bulk adjusted solution temperature reachesapproximately about −38±2 degrees C.; (v) holding the bulk solution atabout −38±2 degrees C. for about six hours or more; (vi) applying vacuumuntil the lyophilization chamber pressure reaches about 50 milliTorr orless; and (vii) maintaining the shelf temperature at about −45±2 degreesC. for about 45 minutes or more after vacuum application.
 5. The methodof claim 4, wherein the primary drying cycle comprises the steps of: (i)raising the shelf temperature to about 0±2 degrees C. at the heatingrate of about 20±2 degrees C. per hour and continue drying under vacuumuntil the product temperature reaches about −3±2 degrees C. or higher;and (ii) raising the shelf temperature to about 25±2 degrees C. andcontinue drying until the product temperature reaches about 20 degreesC. or higher.
 6. The method of claim 4, wherein the secondary dryingcycle comprises the steps of: (i) raising the shelf temperature to about45±2 degrees C. at a rate of about 3 ±2 degrees C./hr and continuedrying until the product temperature reaches about 38±2 degrees C. orhigher; (ii) turning off the vacuum while increasing the chamberpressure with nitrogen; and (iii) when the chamber pressure reachesatmospheric pressure, discontinuing the nitrogen and enclosing thecomposition under a nitrogen atmosphere.
 7. The method of claim 1,wherein the ratio of epoprostenol or a salt thereof to alkalinizingagent is about 1:25 to about 1:200.
 8. The method of claim 1, whereinthe alkalinizing agent is selected from the group consisting ofarginine, lysine, meglumine, N-methyl glucosomine, an amino acid with apKa of 9.0 and above, trisodium phosphates, sodium carbonates, andtetrasodium-EDTA.
 9. The method of claim 1, wherein the bulk solutionfurther comprises (iii) a bulking agent.
 10. The method in claim 9,wherein the bulking agent is selected from the group consisting ofhydroxyl ethyl starch (HES), sorbitol, lactose, dextran, maltose,mannose, ribose, sucrose, mannitol, trehalose, lactose, dextran,cyclodextrin, glycine, and polyvinylpyrrolidine (PVP).
 11. The method ofclaim 9, wherein the bulking agent is present at about 1-10%.
 12. Themethod of claim 1, wherein the epoprostenol salt is epoprostenol sodium.13. The method of claim 1, wherein step (b) comprises adding an organicor inorganic base.
 14. The method of claim 13, wherein the inorganicbase is selected from the group consisting of sodium hydroxide,potassium hydroxide, magnesium hydroxide, and ammonium hydroxide. 15.The method of claim 13, where the organic base is selected from thegroup consisting of aromatic amines and aromatic alcohols.
 16. A methodfor treating a patient suffering from a disease selected from the groupconsisting of cardiovascular disease, atherosclerosis, arteriosclerosis,congestive heart failure, angina pectoris, and hypertension, said methodcomprising the steps of (1) combining an intravenous fluid with aneffective amount of a lyophilized pharmaceutical composition comprising:(a) a unit dose of 0.5 mg or 1.5 mg of epoprostenol or a salt thereof;(b) arginine; and (c) sodium hydroxide, wherein said lyophilizedpharmaceutical composition is formed from a bulk solution having a pH of13 or higher; and (2) administering the resulting intravenous fluid ofstep (1) to a patient in need thereof.
 17. The method of claim 16,wherein step (1) comprises the step of reconstituting the lyophilizedpharmaceutical composition with a first diluent prior to theadministrating step to form a reconstituted solution.
 18. The method ofclaim 17, wherein the first diluent is one of water for injection, 0.9%sodium chloride solution, lactated Ringer's solution, Ringer's solution,sodium carbonate solution, or bicarbonate solution.
 19. The method ofclaim 17, wherein step (1) further comprises the step of diluting thereconstituted solution with a second diluent to form a diluted solution.20. The method of claim 19, wherein the diluted solution ishemocompatible.
 21. The method of claim 17, wherein the reconstitutedsolution is self-preserved.
 22. A method for treating a patientsuffering from a disease selected from the group consisting ofcardiovascular disease or disorder, atherosclerosis, arteriosclerosis,congestive heart failure, angina pectoris, and hypertension, said methodcomprising the steps of (1) combining an intravenous fluid with aneffective amount of a lyophilized pharmaceutical composition comprising:(a) a unit dose of 0.5 mg or 1.5 mg of epoprostenol or a salt thereof;(b) 50 mg of arginine; (c) Mannitol or sucrose; and (d) sodiumhydroxide. wherein said lyophilized pharmaceutical composition is formedfrom a bulk solution having a pH of 13 or higher; and (2) and (2)administering the resulting intravenous fluid of step (1) to a patientin need thereof.
 23. The method of claim 22, wherein step (1) comprisesthe step of reconstituting the lyophilized pharmaceutical compositionwith a first diluent prior to the administrating step to form areconstituted solution.
 24. The method of claim 23, wherein the firstdiluent is one of water for injection, 0.9% sodium chloride solution,lactated Ringer's solution, Ringer's solution, sodium carbonatesolution, or bicarbonate solution.
 25. The method of claim 23, whereinstep (1) further comprises the step of diluting the reconstitutedsolution with a second diluent to form a diluted solution.
 26. Themethod of claim 25, wherein the diluted solution is hemocompatible. 27.The method of claim 23, wherein the reconstituted solution isself-preserved.
 28. The method of claim 22, wherein the intravenousfluid of step (1) is water for injection.
 29. The method of claim 22,wherein the intravenous fluid of step (1) is 0.9% sodium chloridesolution.
 30. The method of claim 22, wherein the intravenous fluid ofstep (1) is 5 mL of water for injection or 0.9% sodium chloridesolution.
 31. The method of claim 22, wherein the administration step(2) comprises administration by injection or infusion.
 32. A method fortreating a patient suffering from a disease selected from the groupconsisting of cardiovascular disease, atherosclerosis, arteriosclerosis,congestive heart failure, angina pectoris, and hypertension, said methodcomprising the steps of (1)(i) reconstituting an effective amount of alyophilized pharmaceutical composition comprising: (a) a unit dose of0.5 mg or 1.5 mg of epoprostenol or a salt thereof; (b) 50 mg ofarginine; (c) Mannitol or sucrose; and (d) sodium hydroxide, in 5 mL ofwater for injection or 0.9% sodium chloride solution to form areconstituted solution, wherein said lyophilized pharmaceuticalcomposition is formed from a bulk solution having a pH of 13 or higher;(1)(ii) diluting the reconstituted solution of step (1)(i) with a seconddiluent to form a diluted solution; and (2) administering the resultingdiluted solution of step (1)(ii) to a patient in need thereof.
 33. Themethod of claim 32, wherein the epoprostenol or a salt thereof isepoprostenol sodium.
 34. The method of claim 33, wherein the seconddiluent in step (1)(ii) is the same diluent as the first diluent used instep (1)(i) to reconstitute the lyophilized composition.
 35. The methodof claim 33, wherein the administration step (2) comprisesadministration by injection or infusion.
 36. The method of claim 16,wherein the intravenous fluid of step (1) is water for injection. 37.The method of claim 16, wherein the intravenous fluid of step (1) is0.9% sodium chloride solution.
 38. The method of claim 16, wherein theintravenous fluid of step (1) is 5 mL of water for injection or 0.9%sodium chloride solution.
 39. The method of claim 16, wherein theadministration step (2) comprises administration by injection orinfusion.
 40. A method for treating a patient suffering from a diseaseselected from the group consisting of cardiovascular disease,atherosclerosis, arteriosclerosis, congestive heart failure, anginapectoris, and hypertension, said method comprising the steps of (1)(i)reconstituting an effective amount of a lyophilized pharmaceuticalcomposition comprising: (a) a unit dose of 0.5 mg or 1.5 mg ofepoprostenol or a salt thereof; (b) 50 mg of arginine; (c) Mannitol orsucrose; and (d) sodium hydroxide, in 5 mL of water for injection toform a reconstituted solution, wherein said lyophilized pharmaceuticalcomposition is formed from a bulk solution having a pH of 13 or higher;(1)(ii) diluting the reconstituted solution of step (1)(i) with waterfor injection to form a diluted solution; and (2) administering theresulting diluted solution of step (1)(ii) to a patient in need thereof.41. The method of claim 40, wherein the epoprostenol or a salt thereofis epoprostenol sodium.
 42. The method of claim 41, wherein theadministration step (2) comprises administration by injection orinfusion.